Aggressive research in genomics, functional proteomics and drug discovery has resulted in a large increase in the number of chemical entities (“leads”) that have a potential for therapeutic activity. The leads are typically pruned in “pre-clinical screening” studies to select promising candidates for final “clinical studies.” Due to the large number of leads to be screened, the pre-clinical screening process has become a bottleneck in the drug discovery process.
During pre-clinical screening, sequential pharmacological transformations of the leads, in conjunction with an organism (e.g., cells, tissues, model animals, etc.,) are evaluated. The evaluation that is typically performed during pre-clinical screening is known as “ADMET” or sometimes “ADME Tox,” which is an acronym for Absorption, Distribution, Metabolism, Excretion and Toxicology. The absorption properties of leads are particularly important, and, as discussed later in this section, are particularly problematic to test.
There are generally two approaches to the pre-clinical screening of leads—in vivo testing and in vitro testing using artificial membranes (immobilized artificial membranes) or cell-based permeability methods. In vivo testing is performed within a living organism, while in vitro testing is performed outside of a living organism. Of these two approaches, in vivo testing provides a more accurate analysis of compound absorption and bio-availability during pre-clinical pharmaco-kinetic studies. Unfortunately, the logistics of animal-based studies makes them extremely expensive and time consuming. Furthermore, in vivo studies cannot provide the speed necessary to support high-throughput screening of drug candidates. Even the recently developed “cassette method,” wherein multiple compounds (about five to ten) are combined and administered to a single animal, cannot provide the desired productivity. (See, J. Berman et al., J. Med. Chem. 40:827-829 (1997); Dietz et al., U.S. Pat. No. 5,989,918.)
Consequently, the focus in high-throughput screening of drug candidates is on various in vitro techniques (and even computer “in silico” modeling methods). Unfortunately, absorption is a difficult process to model and evaluate using in vitro testing. Specifically, absorption deals with the transportation of compounds through live membranes (e.g., tissues, etc.)—a situation that is difficult to re-create outside of a living organism under test conditions. Absorption studies, therefore, have been at the forefront of current drug-discovery efforts. These efforts have been directed at the development of instrumentation and methodologies that will accelerate the pace at which absorption studies can be performed. Specifically, the thrust is to accelerate absorption studies to the speed at which the other steps of the drug discovery process are being conducted.
One of the first methods developed for in vitro absorption studies was the “everted sac” technique (T. H. Wilson & G. Wiseman, J. Physiol. 123:116-125 (1954)). The everted sac is an everted (i.e., mucosal surface turned inside-out) segment of intestine, typically 3 to 5 centimeters in length, that is filled with oxygenated buffer solution (i.e., serosal solution contacting the serosal surface) and tied at both ends with sutures. The everted sac is placed in a similar solution (i.e., mucosal solution) and is incubated at 37° C. with continuous aeration. The compound under test may be added to mucosal or serosal solutions depending on what type of transport is being studied (i.e., mucosa→serosal or serosal→mucosal). After incubation is completed, the concentration of the transported compound is estimated in the solutions on both sides of the intestine and in the intestinal mucosa. This simple, reproducible and inexpensive method is used for studying mechanisms of compound transport through the intestine in various regions, as well as for studying compound metabolism by intestinal mucosa (E. S. Foulkes, Proc. Soc. Exper. Biol. Med. 211:155-162 (1996)).
There are, however, certain disadvantages to the everted-sac technique, including low tissue viability and rapid (thirty minute) onset of histological damage in salt mixtures (R. R. Levine et al., Eur. J. Pharm. 9:211-219 (1970)). Another drawback of the technique is that while the serosal chamber, being a closed system, is appropriate for short-term studies, it might not be suited for the evaluation of molecular kinetics during longer-term studies or when investigating drugs that have a high absorption rate. Furthermore, the everted-sac technique is not suited to high throughput analysis.
Another well-known technique and apparatus for in vitro study of absorption is the “Ussing chamber.” The Ussing Chamber, like the everted intestinal sac, can be used for investigating the transport of molecules and for measuring electrical parameters at specific sites of the intestine, as well as for the evaluation of intestinal metabolism.
Originally developed for measuring the electric potential across frog skin, the Ussing Chamber consists of two chambers—a donor chamber and a receiver chamber. To measure absorption of a compound, a tissue (e.g., whole intestinal tissue or tissue that is stripped from muscular and serosal layers) is placed between the chambers. The chambers are filled with buffer solutions, wherein the compound under investigation is added to the solution within the donor chamber. After incubation (i.e., exposure of the tissue to the compound-containing solution for a certain length of time and at certain conditions of temperature, pH, etc.), aliquots are taken from the receiving chamber or from both chambers, and then analyzed. (H. H. Ussing & K. Zehran. Acta Physiol. Scand. 23:110-127 (1951)). Many modifications of the classical Ussing chamber are used for oral permeability studies. (See, e.g., U.S. Pat. Nos. 4,667,504, 5,183,760, 5,591,636, and 5,599,688.)
There are a variety of drawbacks to the Ussing Chamber. One drawback is the uncertain tissue viability during incubation in simple salt buffers. In particular, it has been demonstrated that after thirty minutes incubation of intestinal mucosa in buffer solution, fifty percent to seventy-five percent of epithelium disappears and, after one hour, total disruption of the epithelial border can occur. (See, Levine et al., Eur. J. Pharm. 9:211-219, (1970)). Even after only twenty minutes of intestinal tissue incubation in a simple salt medium, severe intestinal edema and disruption of epithelium has been observed (M. Mayersohn et al., J. Pharm. Sci. 60:225-230 (1971)).
A second disadvantage of the Ussing Chamber is that it is inappropriate for high-throughput studies. In particular, when using excised tissue samples (as opposed to using cell-culture inserts, e.g., Transwells™), each intestinal strip dissection and mounting takes between two to four minutes (M. Field et al., Amer J. Physiol. 220:1388-1396 (1971)). This makes simultaneous preparation of multiple tissue samples for absorption problematic.
A third disadvantage of the Ussing Chamber is that the ability to obtain samples from the donor chamber and the receiver chamber is limited. In particular, the chambers are filled with fluid (i.e., gas or liquid) that is sampled from each of the chambers as desired. There is no ability to sample fluids from, or deliver fluids to, specific regions within the chambers (e.g., near the tissue sample, etc.).
A relatively new technique, called the “cell-culture” technique, is capable of study absorption at substantially higher throughputs. This technique has already been adopted for automated high-throughput compound screening and optimization. Unlike most in vitro models, the cell culture method does not require the use of the animals, but, rather, uses specific cell lines that are grown in sterile conditions.
One of the key cell lines used for absorption studies is the Caco-2 cell line. Originating from human colon adenocarcinoma, the Caco-2 cells, after confluency, possess many of the functional and morphological characteristics of normal differentiated enterocytes. Multiple studies show that this method provides comparative information on absorption of different drug molecules.
The Caco-2 cells are cultured in a specially-constructed cell-culture plate. The plate consists of an inner well that is disposed within an outer well. The bottom of the inner well is a semi-permeable membrane. The Caco-2 cells are grown to confluency on the semi-permeable membrane. To evaluate transport, a compound is added to a medium above the Caco-2 cells (i.e., the compound is added to the inner well). Uptake of the compound is determined by quantifying the amount of the compound in the medium on the opposite side of the semi-permeable membrane (i.e., in the outer well).
There are a number of drawbacks to the cell-culture technique using Caco-2 cells. One drawback is the cancerous nature of these cells, which might be indicative of altered cellular properties. Furthermore, it takes several weeks to grow the cells. This delays the beginning of absorption tests and increases the risk of bacterial or fungal contamination of the culture (see, L. Barthe et al., Europ. J. Drug Metab. Phatmacokinet. 23:313-323 (1998); A. P. Li et al., High Throughput Screening. Jan. 6-9 (2001)). Caco-2 cells might also be phenotypically unstable and change their enzyme activity and transporter expression with passage number (K. M. Hillgren et al., Med Res Rev 15:83-109 (1995)).
Another major disadvantage of the cell-culture technique is the slow compound absorption rate. Caco-2 cells are between twenty to forty times less permeable than normal human colon cells (P. Artursson et al., Pharm. Res. 10:1123-1129 (1993)). Other studies showed that for mannitol, the rate of permeation is fifty to three hundred and sixty times lower through the cell mono-layer than through the gut (L. Barthe et al., Europ. J. Drug Metab. Phatmacokinet. 23:313-323 (1998)).
Furthermore, the usefulness of Caco-2 cells has been limited because they do not express appreciable quantities of bio-transformation enzymes, which are present in human small-bowel epithelial cells. This drawback was overcome recently by treating Caco-2 cells with vitamin D analog (see U.S. Pat. No. 5,856,189). Some believe, however, that regardless of cell line, “the constraints in the methodology surrounding preparation and use of these cells prevent them from being classified as truly high-throughput screens for absorption” (M. H. Tarbit & J. Berman. Curr. Opin. Che. Biol. 2:411-416 (1998)).
The predominantly manual techniques described above cannot support high-throughput programmable operations, nor provide ease of setup and control functions. Yet, there is a pressing need to implement high-speed screening of compound-to-membrane interactions in many industries and many areas of research. In the context of pharmacological studies, it represents a large area of research in oral, dermal, pulmonary, nasal, buccal, corneal, and vaginal drug absorption.
Notwithstanding the many screening techniques available, a need therefore remains for a device and method that provides at least some of the following advantageous characteristics:                Provides high-throughput screening (“HTS”) with low cost of preparation, operation and maintenance.        Provides high-content screening (“HCS”), which allows multiple in-process sampling and testing that is necessary for time-based kinetic studies.        Enables the use of live tissue as the most relevant substrate for absorption/penetration studies.        Enables the use of artificial membranes of various types when necessary.        Preserves tissue viability by accelerating all steps of the experiment including loading, conditioning and testing.        Provides simultaneous sample loading of all test chambers in order to reduce time and assure valid comparative analysis.        Utilizes small tissue samples, allowing parallel preparation of large numbers of samples from close locations of the same organ, thereby decreasing the effect of absorption gradients due to tissue variation along the small intestine and reducing the required number of donor animals.        Provides selectable and stable testing conditions, which are closely matched in all test chambers of the device.        Maintains, if necessary, an accepted industry-standard number of independent investigative chambers in multiples of 8 or 12 (e.g., 24, 48, 96, etc.).        Maintains in vitro conditions that closely match in vivo conditions, and provides the ability to monitor, alter and “in-process” control these conditions (e.g., temperature, pH, oxygen, etc.).        Has a small physical size for saving bench-top real estate, and is physically adapted for expansion and for the multiplexing of testing chambers.        Provides ease of operation including loading, sampling, cleaning, servicing and maintaining.        